Detection of Potential GDF6 Regulatory Elements by Multispecies Sequence Comparisons and Identification of a Skeletal Joint Enhancer

Genomics 86 (2005) 295 – 305 www.elsevier.com/locate/ygeno

Detection of potential GDF6 regulatory elements by multispecies sequence comparisons and identification of a skeletal joint enhancer

Matthew E. Portnoya, Kelly J. McDermottb, Anthony Antonellisa, Elliott H. Marguliesa, Arjun B. Prasada, NISC Comparative Sequencing Programa,c, David M. Kingsleyd, Eric D. Greena,c, Douglas P. Mortlockb,*

aGenome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA bDepartment of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA cNIH Intramural Sequencing Center (NISC), National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA dDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA

Received 21 March 2005; accepted 24 May 2005 Available online 24 June 2005

Abstract

The identification of noncoding functional elements within vertebrate genomes, such as those that regulate gene expression, is a major challenge. Comparisons of orthologous sequences from multiple species are effective at detecting highly conserved regions and can reveal potential regulatory sequences. The GDF6 gene controls developmental patterning of skeletal joints and is associated with numerous, distant cis-acting regulatory elements. Using sequence data from 14 vertebrate species, we performed novel multispecies comparative analyses to detect highly conserved sequences flanking GDF6. The complementary tools WebMCS and ExactPlus identified a series of multispecies conserved sequences (MCSs). Of particular interest are MCSs within noncoding regions previously shown to contain GDF6 regulatory elements. A previously reported conserved sequence at À64 kb was also detected by both WebMCS and ExactPlus. Analysis of LacZ- reporter transgenic mice revealed that a 440-bp segment from this region contains an enhancer for Gdf6 expression in developing proximal limb joints. Several other MCSs represent candidate GDF6 regulatory elements; many of these are not conserved in fish or frog, but are strongly conserved in mammals. D 2005 Elsevier Inc. All rights reserved.

Keywords: Gdf6; Enhancer elements; Bacterial artificial chromosome; Transgenes; Sequence analysis

The use of comparative sequence analysis for identifying limited. There is often too much aligning sequence between the functional portions of complex genomes has great pairs of closely related species, forcing the use of somewhat potential for facilitating the detection of noncoding functional arbitrary thresholds (e.g., >70% identity across 100 bp). elements. Pair-wise sequence alignments and comparisons Meanwhile, there is often too little aligning sequence can be used for this purpose, for example, by simply assessing between pairs of more distantly related species, especially the percentage sequence identity across windows of a defined within noncoding regions. To overcome the limitations of size [1]. Such approaches have proven effective at identifying simple pair-wise analyses, approaches for performing multi- cis-acting regulatory sequences, including those associated species sequence comparisons have been developed [5–7]; with developmentally regulated vertebrate genes, which tend these provide a more powerful means of identifying the most to have multiple, distantly located regulatory elements [2–4]. highly conserved genomic regions. It is thus of great interest However, the use of pair-wise sequence comparisons is to apply these new approaches for studying genomic regions thought to contain complex, cis-acting regulatory elements. * Corresponding author. Fax: +1 615 343 8619. GDF6 is a member of the BMP (bone morphogenetic E-mail address: [email protected] (D.P. Mortlock). protein) gene family, a group of genes that encode secreted

0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2005.05.003 296 M.E. Portnoy et al. / Genomics 86 (2005) 295–305 signaling factors. Like other BMP genes, GDF6 is Using the previously established sequence of this BAC as a expressed in many anatomical locations during embryonic reference, we have generated (¨2.7 Mb) or obtained (¨1.5 development, including numerous skeletal joints [8,9], and Mb) sequences of the orthologous genomic regions from 13 it is required for normal formation of limb, ear, and skull additional vertebrates (Fig. 1a). A previous PipMaker joints [9]. These findings are consistent with the known analysis of the mammalian GDF6 sequences has been roles of BMP members in mediating many developmental described [16]. To examine the degree of noncoding processes, such as the regional control of bone growth and conservation in other vertebrates, we obtained sequences shape [8,10,11] and soft tissue development [12,13].As from additional species and performed MultiPipMaker with other developmentally important genes, the localized analysis on the entire data set. For nine species (chimpan- effects of BMPs seem to be largely controlled by modular zee, baboon, cow, pig, cat, dog, rat, platypus, and ze- arrangements of cis-acting regulatory sequences that control brafish), appropriate BACs were isolated and sequenced the expression of BMP genes in specific anatomical as previously described [16,17]. For four species (human, locations. These regulatory sequences can reside far away chicken, Fugu, and Xenopus), the orthologous sequence from the gene [14,15]; for example, mouse bacterial was obtained from the whole-genome sequence assembles artificial chromosome (BAC)-transgene studies revealed available at the UCSC Genome Browser (http://genome. that regulatory sequences are distributed across a region of ucsc.edu/) [18]. more than 100 kb encompassing the GDF6 gene and that Pair-wise alignments were generated between the mouse these elements mediate GDF6 transcription in limb joints, reference sequence and each of the other species’ sequences digits, retina, genitalia, laryngeal cartilages, skull bones, and using BLASTZ [19], with the results visualized using other tissues [4]. MultiPipMaker [20]. Sequence coverage of the region To localize and identify further individual GDF6 cis- immediately encompassing Gdf6 was nearly complete for acting regulatory elements, we performed extensive multi- most species, with minor exceptions (Fig. 1b). The two species sequence comparisons. Previously, we performed GDF6 exons are highly conserved across vertebrates, as are pair-wise comparisons using PipMaker to indicate con- several intronic noncoding regions (Fig. 1b). As reported served GDF6 sequences [16]. Here, we describe the use of previously [16] much of the noncoding DNA flanking the two multispecies comparative approaches for detecting GDF6 gene is conserved among mammals. However, a highly conserved regions in and around GDF6. These more limited set of flanking regions is also conserved in methods detect a developmentally regulated GDF6 chicken and Xenopus. The mouse–zebrafish and mouse– enhancer that can direct gene expression in proximal limb Fugu alignments in regions flanking GDF6 appear to be joints in vivo. Our findings suggest that multispecies spurious (i.e., related to simple repeat-like sequences; data conserved sequence (MCS) analysis may be a sensitive not shown); however, true mouse–fish conserved ortholo- approach for detecting other GDF6 regulatory elements. gous sequences were found within the GDF6 exons and intron.

Results WebMCS and ExactPlus analyses

Multispecies sequences and alignments Since MultiPipMaker essentially displays separate pair- wise comparisons, we then performed two multispecies The 209-kb mouse BAC clone RPCI23-117O7 contains comparative analyses (WebMCS and ExactPlus) to priori- the entire Gdf6 gene and extensive flanking regions [4]. tize conserved regions by assessing multiple alignments

Fig. 1. Multispecies comparative sequence analysis of the genomic region encompassing GDF6. (a) Venn diagram showing the major cladistic relationships of the vertebrate species whose sequences were analyzed. (b) A low-resolution overview of MultiPipMaker analysis of the GDF6-containing sequences from 14 species, with the mouse sequence used as the reference. The horizontal colored bars at the top of the overview plot (red, yellow, green, blue, and magenta) indicate positions of five contiguous genomic regions in mouse that were previously identified by BAC-transgene analysis to contain different subsets of tissue- specific Gdf6 regulatory enhancers [4]. The relative positions and orientations of mouse Gdf6 and two pseudogenes (Uqcrb and Gapdh) are indicated, as is the 2.9-kb interval previously shown to contain a proximal joint enhancer (PJE) [4]. For each species, portions of the mouse reference sequence that align to that species’ sequence are indicated by green and red bars (reflecting regions with >50 or >75% identity with the mouse sequence, respectively). Gray bars indicate known gaps in the sequence data that are greater than 1 kb. (c) UCSC Genome Browser-based view depicting the positions of MCSs around the mouse Gdf6 gene (mm4/NCBI build 32, October 2003; shown is the interval chr4:9,641,000–9,850,732, which corresponds to bases 50,001–209,733 of mouse BAC RP23-117O7; GenBank No. AC058786). The top 10 tracks (labeled ‘‘EP’’) depict the positions of MCSs detected with ExactPlus; these are labeled using the format ‘‘EP: #-#-#,’’ in which the three numbers indicate the initial seed length (in bases), minimum number of species whose sequence needs to match the initial seed region, and minimum number of additional species’ sequences used to extend the initial match in either direction (see Results for details). The two WebMCS tracks depict MCSs corresponding to the top 5% (WebMCS-95) and 2% (WebMCS-98) most conserved sequence. The ‘‘Transgenes’’ track shows the positions of the previously tested 2.9-kb PJE fragment [4] and the 440-bp fragment tested in this study (see Figs. 3 and 4). The ‘‘Known Genes’’ track (top) shows the positions of Gdf6, a Riken clone transcript that probably originates from the Uqcrb pseudogene [4], and a LINE-containing transcript (GenBank No. U156547). The ‘‘Spliced ESTs’’ and ‘‘Non-Mouse mRNAs’’ tracks show data from the UCSC Genome Browser for positions of spliced mouse ESTs and regions having protein-coding homology to nonmouse mRNAs, respectively. The ‘‘RepeatMasker’’ track displays the locations of repetitive elements identified by the RepeatMasker program. M.E. Portnoy et al. / Genomics 86 (2005) 295–305 297 simultaneously. Both programs are designed to detect WebMCS uses a previously described binomial-based MCSs, although the underlying algorithms used by each approach [6] to derive a ‘‘conservation score’’ for each base are quite distinct. Note that we use the term MCS to refer to in a reference sequence by analyzing windows across a a conserved region detected by multispecies sequence multispecies sequence alignment. WebMCS can be imple- comparisons, regardless of the specific method used to mented to detect different amounts of conserved sequence; perform those comparisons. Both WebMCS and ExactPlus for example, WebMCS-95 and WebMCS-98 identify the top use the same MultiPipMaker-generated multisequence 5% and 2% mostly highly conserved bases in the reference alignment as input and generate similar output files that sequence, respectively. ExactPlus finds small blocks of can be uploaded to the UCSC Genome Browser for bases (or ‘‘seeds’’) of a designated size such that each base visualization (Fig. 1c). in a block is identical across a defined minimum number of 298 M.E. Portnoy et al. / Genomics 86 (2005) 295–305

Table 1 MCS detection with WebMCS MCS detection No. Total Avg. MCS No. of No. of Coding bases Coding Noncoding Sensitivity Specificity method MCSs MCS length coding noncoding overlapping bases MCS basese of detecting of detecting detected bases (bases) MCSsa MCSsb MCSsc missedd coding basesf coding basesg WebMCS-95 153 10,488 69 8 145 1111 251 9377 0.816 0.106 WebMCS-98 58 4201 72 9 49 829 533 3372 0.609 0.197 a Number of MCSs that overlap protein-coding sequence by at least 1 base. Note that the only protein-coding sequence in the region resides in GDF6 exons 1 and 2. b Number of MCSs that do not overlap protein-coding sequence by at least 1 base. c MCS bases that overlap the 1362 bases of GDF6 coding bases. d GDF6 coding bases not overlapping MCSs. e MCS bases not overlapping GDF6 coding sequence (total MCS bases – coding bases). f Coding bases overlapping MCSs/(coding bases overlapping MCSs + coding bases missed). g Coding bases overlapping MCSs/total MCS bases. species (Antonellis et al., manuscript in preparation). The To assess qualitatively how WebMCS and ExactPlus seeds can then be extended in either direction based on perform on the GDF6 data set, we ran each with different identity across a separately defined minimum number of input parameters and quantified the number of MCSs and species (see Materials and methods for details). The amount of conserved sequence detected in each case. extension step was designed in an attempt to detect ancient, WebMCS-95 and WebMCS-98 detect 153 (average size of strongly conserved sequences that could represent the core 69 bases) and 58 (average size of 72 bases) MCSs, of a larger functional element. For example, in regulatory respectively, within the roughly 209,000-base multise- elements such as enhancers, core transcription factor-bind- quence alignment (Table 1). Of these, 8 and 9 MCSs, ing sites may be highly conserved while flanking sequences respectively, overlap the two GDF6 protein-coding exons may have evolved considerably. (Fig. 2). Not surprisingly, ExactPlus detects different

Fig. 2. MCSs in the immediate region spanning the GDF6 transcription unit. An expanded 21-kb region from Fig. 1c highlights the interval spanning from 2 kb upstream to 1 kb downstream of the GDF6 gene in the UCSC Genome Browser-based view. WebMCS-98 and selected ExactPlus data tracks are shown below the GDF6 gene structure (see Fig. 1 for details). Arrows indicate MCSs located outside the GDF6 exons (see text for details). Individual MCSs in certain tracks have been labeled with ‘‘L_#’’ to indicate the length of the MCS in bases. In the WebMCS-98 track, the light and dark shading of MCSs is used to represent MCSs that do and do not overlap, respectively, a GDF6 exon by at least 1 base. M.E. Portnoy et al. / Genomics 86 (2005) 295–305 299 numbers of MCSs depending upon the parameters used for in the GDF6 intron (Figs. 1c and 2, Table 2), indicating initial seed size and for the minimum number of species ancient conserved sequences. This is consistent with the required for seeds and extended bases. For example, using recent findings that noncoding sequences that are highly low-stringency parameters (6-base seeds initiated with 6 conserved between mammals and fish often reside near species and extended in 6 species, designated as 6-6-6), developmentally regulated genes [21,22]. ExactPlus detects 1621 MCSs (Fig. 1c and Table 2). If the seed length is increased to 10 bases and the number of initial Conserved sequences in the GDF6 promoter, intron, and 3V species is increased to 9 (10-9-6), only 136 MCSs are UTR detected (Fig. 1c and Table 2). Interestingly, using the latter parameters, nearly all (98.8%) of the MCS bases detected by The above results indicate the presence of several highly ExactPlus overlap those detected by WebMCS-95 (Table 2). conserved sequences close to the GDF6 gene that may be As expected, increasing the ExactPlus initial seed length candidates for ancient cis-acting regulatory elements. to 25 or 50 bases greatly reduces the number of identified Indeed, in zebrafish and Xenopus, Gdf6 is transcribed in MCSs; however, greater than 97% of the resulting MCS the dorsal retina and dorsal neural tube [23,24], and similar bases overlap with those detected by WebMCS-95 (Fig. 1c patterns of mouse Gdf6 expression were implied by BAC- and Table 2). Several distal flanking regions are still transgene experiments [4]. Therefore we scrutinized the area detected by ExactPlus using a 50-base seed, including: (1) proximal to GDF6 in more detail. Fig. 2 depicts the a region at À63.2 kb within the previously mapped limb positions of MCSs detected by WebMCS and ExactPlus joint regulatory segment [4], (2) a region at À1.0 kb, (3) two within the region immediately encompassing the GDF6 regions in the GDF6 intron, (4) portions of the GDF6 exon transcription unit. At À1.0kbrelativetotheGDF6 2 coding region and the 3V UTR, and (5) two regions at translation start site an MCS was detected by WebMCS- +53.7 and +57.7 kb (Figs. 1c, 2, and 3; note that all 98 and ExactPlus (using two sets of ExactPlus parameters, coordinates are given with respect to the GDF6 5V end). 10-9-6 and 50-6-9; this MCS is labeled ‘‘L_69’’ in the EP: With sequences from 9 eutherian mammals included in our 50-6-9 parameter track within Fig. 2). WebMCS-98 and data set, the initial requirement of 10 species’ sequences to ExactPlus (with 25-6-2 parameters) also detect an MCS just match the seed might discriminate between eutherian- upstream of the mRNA 5V end (the leftmost L_38 in specific conserved sequences and more ancient conserved WebMCS-98 and L_74, respectively), suggesting conserva- sequences. For example, a conserved region (approximately tion within the GDF6 promoter; this region was not detected 75 bases) roughly 1 kb upstream of the first GDF6 exon is by ExactPlus using a shorter initial seed length and a larger nearly identical in all eutherian mammals but not in any number of initial and extension species (EP: 10-9-6). Both noneutherian species (Figs. 1b, 1c, and 2); this may reflect a of these regions are within a genomic interval shown to have eutherian-specific regulatory sequence(s). neural tube and brain enhancer activity affecting GDF6 Increasing the ExactPlus minimum-species number at expression [4]. initial seed to 12 results in the identification of regions that Three notably large ExactPlus-detected MCSs reside are conserved in all mammals and at least one of Xenopus, within the GDF6 intron (labeled A, B, and C in Fig. 2). Fugu, or zebrafish. Interestingly, this identifies two MCSs These may function to regulate GDF6 expression in dorsal

Table 2 Relationship of MCSs detected by ExactPlus and WebMCS-95 MCS detection No. MCSs Total No. MCSs MCS bases WebMCS-95 WebMCS-95 method detected MCS overlapping with overlapping with sensitivityc specificityd bases WebMCS-95a WebMCS-95b WebMCS-95 153 10,488 N/A N/A N/A N/A ExactPlus 6-6-6 1621 16,002 575 7787 0.487 0.742 ExactPlus 6-9-6 320 5089 275 4660 0.916 0.444 ExactPlus 10-9-6 136 3118 133 3080 0.988 0.294 ExactPlus 25-6-2 42 2973 42 2890 0.972 0.276 ExactPlus 25-9-6 21 901 21 901 1.000 0.086 ExactPlus 50-6-9 8 543 8 543 1.000 0.052 ExactPlus 6-10-2 114 4348 110 4140 0.952 0.395 ExactPlus 10-10-2 57 2844 57 2823 0.993 0.269 ExactPlus 25-10-2 7 637 7 637 1.000 0.061 ExactPlus 10-12-2 8 511 8 511 1.000 0.049 N/A, not applicable. a Number of ExactPlus MCSs that overlap with WebMCS-95 MCSs by at least 1 base. b Number of ExactPlus MCS bases that overlap with WebMCS-95 MCS bases. c Fraction of ExactPlus MCS bases that overlap with WebMCS-95: MCS bases overlapping with WebMCS-95 MCSs/total ExactPlus MCS bases. d Fraction of ExactPlus MCS bases also detected by WebMCS-95: MCS bases overlapping with WebMCS-95/10,488. 300 M.E. Portnoy et al. / Genomics 86 (2005) 295–305

Fig. 3. Highly conserved regions and potential transcription factor binding sites in the PJE region. (a) UCSC Genome Browser-based view highlighting the positions of MCSs in a 1.2-kb interval within the PJE region (see Figs. 1b and 1c for orientation). Individual MCSs in certain tracks have been labeled with ‘‘ L _#’’ to indicate the length of the MCS in bases. ‘‘2.9Kb_tg’’ and ‘‘440bp_tg’’ represent the locations of the 2.9-kb transgene [4] and 440-bp transgene (PJE- 440; see Results), respectively. Arrows indicate locations of three potential Pbx1/Hox binding motifs (see Results). (b and c) Putative transcription factor binding motifs within specific MCSs (indicated in (a) by asterisks). Coverage across the motif regions is shown for selected MCSs that were detected using different parameters, as indicated. retina or genitalia [4]. In addition to the MCSs overlapping minigene cassette in elbow, knee, shoulder, and hip joints the coding portion of exon 2, an MCS was also found in the [4]. The presumed enhancer in this fragment was called 3V UTR just upstream of the noncanonical ATTAAA the PJE (proximal joint element). Two potential hetero- polyadenylation signal. This conserved sequence may play dimeric binding sites for Pbx1/Hox transcription factors a role in the posttranscriptional regulation of GDF6 mRNA. [26] have been detected in this region, consistent with a We were unable to detect evidence for an RNA secondary role for this sequence in proximal limb patterning structure within this MCS (e.g., long hairpins or stem– [16,27]. loops). Interestingly, a highly conserved sequence has been We therefore examined WebMCS and ExactPlus results found in the 3VUTR of another BMP family gene, BMP2, for this region. Both methods detect multiple MCSs in the where it may regulate mRNA stability [25]. 2.9-kb region (Fig. 3). Both programs also identify a highly conserved region of approximately 300 bp within the 2.9-kb Conserved sequences regulate GDF6 expression region found to contain the PJE (Fig. 3a). This conserved region is present in all mammalian sequences (except pig, In the embryonic limb bud, GDF6 is transcribed in a for which sequence coverage is lacking in this region), but stripe-like pattern that marks the locations of interzones, not Fugu, zebrafish, or Xenopus (Fig. 1b). Thus, this likely histologically distinct regions that give rise to skeletal reflects a mammal-specific conserved element. There is also joints [8,9]. Previous transgenic studies indicated that a a less conserved region several hundred bases downstream region far upstream (>60 kb) of GDF6 is required for its that is detected by WebMCS-95 and ExactPlus with low- expression in proximal limb joints during embryonic stringency parameters (6-6-6); however, this region is not development [4]. Furthermore, a 2.9-kb fragment from detected by WebMCS-98 or ExactPlus with higher strin- this region can drive expression of a LacZ-containing gency parameters (Fig. 3a). M.E. Portnoy et al. / Genomics 86 (2005) 295–305 301

Within the highly conserved approximately 300-bp the Lef1/TCF factors function in the Wnt ligand signaling region reside several stretches of high conservation, as pathway [30,31] and that Wnt14 has been proposed to be a detected by ExactPlus. One block of 56 bp was detected key regulator of early joint development [32,33]. These data using either 25:9:6 or 50:6:9 parameters; this may reflect a suggest that Hox and Pbx factors may interact with the Wnt core enhancer element (Fig. 3b). Closer inspection of this signaling pathway to regulate joint-specific gene expression. block revealed that it overlaps with the second of the two To test if the highly conserved L_56 region indicates the previously reported Pbx1/Hox binding motifs [16]. A third core of a cis-acting sequence that enhances GDF6 expression Pbx1/Hox binding motif was also found. Interestingly, this in the proximal limb joint, a 440-bp segment encompassing Pbx1/Hox motif is flanked by two CTTT(T/A)A(T/A) L_56 was subcloned into an Hsp68 promoter–LacZ motifs, which are similar to the consensus Lef1/TCF1 minigene construct [4,34]. Transgenic mouse embryos binding site [28,29]. WebMCS-98 detects one long (L_221) containing this construct (PJE-440) were analyzed midg- and one short (L_35) MCS in this region. While the former estation for LacZ expression, with the results summarized in contains the three Pbx1/Hox motifs, the latter contains three Fig. 4 and Table 3. Of seven independently generated imperfect matches to the Lef1/TCF consensus sequence transgenic embryos, several had staining in the eye, which (Fig. 3c). These results are particularly striking given that we have found to be a common expression artifact of the

Fig. 4. A conserved sequence located 64 kb upstream of GDF6 functions as a transcriptional enhancer in proximal limb joints. The PJE-440 construct (containing a highly conserved 440-bp portion of the PJE region cloned into a heat-shock promoter/LacZ reporter vector) was injected into one-cell mouse embryos. Transgenic embryos were harvested at 14.5 dpc, cut in half down the midline, stained with X-gal, and cleared with glycerol for imaging. (a) A representative embryo is shown, with white arrowheads indicating LacZ expression in shoulder, elbow, and knee joints. The hip joint was also stained but not retained in the photographed half of the embryo after dissection (data not shown). LacZ expression in the eye is a cryptic effect of the transgene vector and is likely not associated with the 440-bp sequence (see Results). The faint staining in the brain is due to background (i.e., nontransgenic) h-galactosidase activity. (b) Close-up view of the upper arm, showing shoulder and elbow joint staining at the ends of the humerus. (c) Section through the elbow of a transgenic embryo reveals that staining is specific to cells in the humeroradial and humeroulnar joints. (d) Close-up view of the knee joint showing two separate major domains of staining, plus a faint area of more proximal staining near the future patella. (e) Schematic drawing of the two major staining domains relative to the femur and tibia. (f –i) Near-adjacent serial sections through the knee of a transgenic embryo. hu, humerus; ra, radius; ul, ulna; fe, femur; ti, tibia. 302 M.E. Portnoy et al. / Genomics 86 (2005) 295–305

Table 3 encompassing GDF6 in multiple additional vertebrates. PJE-440 transgene expression in mouse embryos Two analytical methods, WebMCS and ExactPlus, were Embryo Proximal Ectopic expression used to analyze these multispecies sequences, allowing the limb joints identification of a large set of MCSs. The majority of these 1 +++ Some phalangeal joints, limb reside within noncoding regions. More detailed functional tendons, brain, neural tube, analysis of one MCS region identified an enhancer (the PJE) stomach 37 +++ Retina that mediates GDF6 expression in limb joints. This has 38 – Widespread ectopic refined the PJE location from 2.9 kb to 440 bp (Figs. 3 and staining 4), although both WebMCS and ExactPlus suggest the 65 + critically conserved core of the PJE is closer to 300 bp (Fig. 69 – Brain (weak) 3; see below). 72 +++ Retina 82 ++ Retina, forebrain, neural tube Our findings indicate that ExactPlus and WebMCS 88 +a None represent complementary approaches for identifying con- a Weak staining in elbow joint only. served sequences, with degree of overlap depending on input parameters. Though virtually all MCSs detected by highly stringent implementation of ExactPlus tend to over- Hsp68 –LacZ vector backbone (D.P.M., D.M.K, unpub- lap with WebMCS elements, when smaller seeds and lished observations; Catherine Guenther, personal commu- matching-species parameters are used some important nication) (Fig. 4a and Table 3). However, five of the differences are notable. For example, ExactPlus is more embryos showed strong LacZ expression in the nascent likely to detect small sequence blocks (e.g., on the scale of elbow, knee, shoulder, and hip joints (Figs. 4a, 4b, and 4d), transcription factor-binding sites) than WebMCS, which in a pattern essentially identical to that previously observed analyzes sequences in 25-base windows [6].Wealso with the 2.9-kb segment [4]. This indicates that the 440-bp suggest that ExactPlus may be a useful alternative to fragment can function as a modular regulatory element methods that search for consensus motifs for transcription capable of activating a heterologous promoter specifically in factor binding sites. For example, the potential nonconsen- proximal limb joints. sus TCF/Lef binding sites depicted in Fig. 3 may fall into Histological sections of PJE-440-containing embryos this category. At the same time, regulatory elements often confirmed that the LacZ expression is restricted to the contain multiple transcription factor-binding sites that joints (Figs. 4c and 4f–4i), generally within the articular function redundantly, with the amount of sequence con- cartilage and also in intervening cells between the cartilage servation at any one site being variable among species. In elements and at a developmental stage prior to cavitation of these situations, WebMCS should be particularly effective the joint cavity. Interestingly, in the knee joint, two arc- since it assesses conservation with sliding windows and shaped domains of LacZ expression are apparent. Section- does not require 100% sequence identity. In other words, ing revealed that these domains curve around the lateral and WebMCS probably tolerates plasticity better than ExactPlus. medial condyles of the distal femur where it articulates with The amount and distribution of conserved sequences the tibia (Figs. 4d–4i). We also tested a larger construct that, across the GDF6 genomic region are broadly similar among in addition to the 440-bp segment, contains the less well the nonrodent eutherian mammals (see Fig. 1b). A very conserved region about 400 bp downstream (see EP: 6-6-6 different pattern is seen for platypus (a noneutherian and WebMCS-95 blocks, right side of Fig. 3a). This larger mammal), with conservation largely confined to the most construct confers LacZ expression in proximal limb joints in stringently defined MCSs. Comparisons with the ortholo- a pattern indistinguishable from that of the PJE-440 gous chicken sequence reveal similar findings, though fewer construct (data not shown). While the functional relevance regions of conservation are noted. Conservation between the (if any) of this less well conserved region is unclear, the mouse and the nonamniote species (Xenopus tropicalis, 440-bp region seems to contain the GDF6 PJE. Fugu, and zebrafish) is mostly confined to GDF6 coding sequences and a minimal number of noncoding regions near GDF6 and was well detected by both WebMCS and Discussion ExactPlus. Across the GDF6 region, the platypus sequence appears to be the most effective for identifying noncoding Previous human and mouse sequence comparisons MCSs, particularly for segments flanking the gene. These suggested the presence of numerous conserved noncoding results are consistent with a recent larger study investigating regions within and flanking the GDF6 gene [4,16]. These the utility of noneutherian mammal (marsupial and monot- regions represent tantalizing candidates for serving as cis- reme) sequences for identifying conserved genomic regions acting regulatory elements that mediate the complex [35]. expression of GDF6 [8,9]. Here, we report an extension MCS analysis indicates the PJE is well conserved among of those studies that has involved the generation and amniote species. The discovery of the PJE also provides comparison of the sequence of the genomic region new insights into potential roles for GDF6 in the patterning M.E. Portnoy et al. / Genomics 86 (2005) 295–305 303 of embryonic limb joints. GDF6 transcription in the (e.g., limb joint adaptations compatible with terrestrial embryonic elbow joint has been previously documented mobility). Further transgenic experiments to compare the [4,9]. However, because the GDF6 mRNA is transcribed at regulatory functions of candidate GDF6 enhancers in low levels we have found it difficult to characterize its mammals, frog, and fish will be useful for investigating expression in the other proximal limb joints. In contrast, the this possibility. more sensitive transgenic LacZ assay has proven valuable for characterizing the function of the GDF6 PJE. Taken together with our comparative sequence analyses, the LacZ Materials and methods expression data strongly suggest a conserved role for GDF6 in proximal joint patterning. Interestingly, in the knee joint BAC clones and sequences PJE-regulated transgene expression was restricted to sub- regions within the joint cavity. Given the ability of GDF The Gdf6-containing mouse BAC RPCI23-117O7 proteins to stimulate chondrogenesis [36–38], the trans- sequence (GenBank No. AC058786) served as the reference genic expression pattern suggests that GDF6 regulates for comparative analyses and corresponds to UCSC growth of the adjacent femoral condyles. Genome Browser coordinates chr4:9,641,000–9,850,732 MCS analysis was useful for stimulating hypotheses for (mm4/NCBI build 32, October 2003). Orthologous human candidate PJE-binding factors. The PJE contains putative and rat genomic sequences were retrieved from the binding sites for Pbx1/Hox heterodimers, factors that both respective genome-wide data sets [16,42,43]. Minimally pattern proximal limb tissue [27]. Studies in Drosophila overlapping GDF6-containing BACs from rat, chimpanzee, suggest that Hox-binding regulatory modules typically baboon, cow, pig, cat, dog, platypus, and zebrafish were require additional inputs from interacting signaling path- identified [17] and sequenced as part of the NISC ways [39]. We hypothesize that the PJE integrates positional Comparative Sequencing Program [5] (www.nisc.nih.gov). information provided by the Pbx/Hox factors with other Accession numbers for all of the above BAC sequences signaling pathways that specify skeletal joints. Lef1/TCF were previously reported [16] except for zebrafish BAC factors function in the Wnt signaling pathway, raising the CH211-216G21 (GenBank No. AC139623). Sequences intriguing possibility that Hox and Pbx patterning factors were finished to a grade that is of intermediate quality interact with Wnt proteins to specify expression in devel- between phase I (full shotgun) and phase III (contiguous, oping joints. Indeed, Wnt14 is thought to direct early near perfect); this is called ‘‘comparative-grade finished synovial joint development [32]. Further testing should sequence’’ and is an enhanced version of phase II finished determine if Pbx, Hox, and/or TCF/Lef proteins bind the sequence [44]. Additionally, sequence from an orthologous PJE in vitro or in vivo. We also note that the long tracts of Gdf6-containing X. tropicalis BAC (CH216-129O13, Gen- sequence conservation in the PJE are not easily explained by Bank No. AC147884) was identified and downloaded from the detected Pbx/Hox and Lef1/TCF binding sites, so the the DOE/JGI Web site (http://genome.jgi-psf.org/xenopus). binding of additional transcription factors is probably Additional orthologous sequences were obtained from the important for PJE function. Further analysis of ExactPlus UCSC Genome Browser as follows: (1) chimpanzee, used data may help characterize binding sites for such factors. to fill in gaps in the chimpanzee BAC sequence (NCBI In addition to skeletal joints, GDF6 is transcribed in the chimpanzee draft genome sequence build 1, November developing skull, larynx, and digits, and previous BAC- 2003; chr7:99,408,595–99,511,759); (2) chicken (build transgenic data suggest that GDF6 is expressed in neural galGal2, Chicken Genome Sequencing Consortium, Febru- tube, retina, teeth, and other tissues [4]. Interestingly, these ary 2004; chr2:124,800,000–124,950,000; also see structures show varying degrees of morphological diversity www.genome.wustl.edu/projects/chicken); (3) Fugu rubri- across vertebrates. Thus, GDF6 enhancers might be pes (Fugu build fr1, August 2002, chrUn:140,629,874– expected to vary considerably with respect to sequence 140,862,748; International Fugu Genome Consortium; also conservation. Studies of other developmentally regulated see www.fugu-sg.org). genes have shown that the more conserved the sequence between highly divergent species (for example, mammals Comparative analysis and MCS tools and fish), the more likely it is to be functionally important [21,22]. However, other studies indicate that many sequen- Multispecies sequence comparisons were performed using ces that serve to regulate gene expression are not conserved MultiPipMaker [20], as described [16]. MCSs were detected between mammals and fish [40,41]. At least three GDF6 using ExactPlus (Antonellis et al., manuscript in prepara- noncoding MCSs are conserved between mouse and frog tion; http://research.nhgri.nih.gov/projects/exactplus)or and/or fish, making them candidates for being ancient WebMCS [6] (http://research.nhgri.nih.gov/MCS). Briefly, GDF6 enhancers. Other GDF6-associated MCSs, such as ExactPlus finds small blocks of bases (or ‘‘seeds’’) of a the PJE, are not found in fish or frog. We speculate that this designated size such that each base in a block is identical reflects the evolution of new GDF6 regulatory capabilities across a defined minimum number of species in the Multi- correlating with morphological adaptations in amniotes PipMaker alignment. The seeds can then be extended in either 304 M.E. Portnoy et al. / Genomics 86 (2005) 295–305 direction in a base-by-base fashion, on the condition that each with isopropanol/paraffin, and embedded in paraffin over- extended base must be identical in a defined minimum night. Sections (10–12 Am) were cut using a microtome, number of species (note that the minimum number of species transferred to glass slides, dewaxed briefly with xylene, can be different in the initial seed and subsequent extension and counterstained with eosin prior to microscopic steps). The extension steps attempt to detect the presence of imaging. ancient, strongly conserved sequences that represent the core of a larger functional element. For example, in regulatory elements such as enhancers, the core transcription factor- Acknowledgments binding sites can be highly conserved, while the immediate flanking sequences may have evolved considerably. For all We thank numerous people associated with the NISC analyses, the presumptive mouse Gdf6 transcription start site Comparative Sequencing Program, in particular Robert was assumed to be the 5Vend of a Gdf6 mRNA sequence Blakesley, Gerry Bouffard, Jennifer McDowell, Baishali (GenBank No. AJ537425). Maskeri, Nancy Hanson, the many dedicated mapping and sequencing technicians, and other staff. We also thank Laura Transcription factor site analysis Selenke and Lissett Ramirez for expert technical assistance and Karen Deal, Maureen Gannon, Anna Means, and Laura MacVector software was used to scan the mouse PJE Wilding for generously sharing equipment and advice. We sequence for previously reported consensus binding sites for also thank Ronald Chandler for helpful discussions. Kelly J. Pbx1/Hox heterodimers [26] and Lef/TCF. The Pbx1/Hox McDermott was supported by NIH Genetics Training Grant binding sequence ATGATTTA(C/T)GAC was previously 1T32GM62758-03. David M. Kingsley was supported by determined using heterodimers of Pbx1 protein with Hox NIH Grant 5R37AR042236-12. Douglas P. Mortlock was proteins of Hox groups 9 and 10 [26]. The Lef/TCF binding supported by NIH Grant 1R01HD47880-01. Transgenic consensus used was CTTTG(A/T)(A/T), to reflect both the mice were generated by the Vanderbilt University Trans- reported TCF1 binding site CTTTGTT [29] and the reported genic and ES Cell Shared Resource, which is supported by Lef1 binding site CCTTTG(A/T)(A/T) [28]. Consensus the Vanderbilt Cancer, Diabetes, Kennedy, and Vision binding sites were scanned using the MacVector Nucleic Centers. Acid Subsequence analysis tool set to permit mismatches anywhere within the consensus sequence. 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